qPCR data QC

library(tidyverse)
library(magrittr)
library(gt)
library(patchwork)
library(SummarizedExperiment)
library(tidySummarizedExperiment)

tmp <- fs::dir_map("R scripts/", source)
tmp <- fs::dir_map("../VIBRANT-99-utils/R/", source)
rm(tmp)

theme_set(theme_light())

The purpose of this document is to perform various quality check on the qPCR data generated on “weekly swabs” from the VIBRANT clinical study. “Weekly swabs” are swab samples collected at their weekly in-clinic visits.

Loading the data

qpcr_dir <- str_c(get_data_dir(), "03 qPCR/")
file <- "VIBRANT_qPCR_data_merged_250611.csv"
cat("We load the file", file, " (15 LBP strains)\n\n")

We load the file VIBRANT_qPCR_data_merged_250611.csv  (15 LBP strains)

qpcr_LBP <- read_csv(str_c(qpcr_dir, file))

file <- "VIBRANT_16SqPCR_250616.csv"
cat("\n\nAnd the file ", file, " (16S)\n\n")

And the file  VIBRANT_16SqPCR_250616.csv  (16S)

qpcr_16S <- read_csv(str_c(qpcr_dir, file))

rm(qpcr_dir, file)

The data is provided in long format:

qpcr_LBP |> glimpse()

Rows: 54,810
Columns: 21
$ Data_row                 <dbl> 2275, 2276, 2277, 1603, 1604, 1605, 2272, 227…
$ Well                     <chr> "E14", "E15", "E16", "E14", "E15", "E16", "E1…
$ Fluor                    <chr> "HEX", "HEX", "HEX", "Cy5", "Cy5", "Cy5", "FA…
$ Content                  <chr> "Unkn-23", "Unkn-23", "Unkn-23", "Unkn-23", "…
$ Replicate                <dbl> 23, 23, 23, 23, 23, 23, 23, 23, 23, 23, 23, 2…
$ Sample                   <chr> "C5", "C5", "C5", "C5", "C5", "C5", "C5", "C5…
$ Cq                       <dbl> 30.91797, 30.97083, 30.74273, NA, NA, NA, NA,…
$ `Starting Quantity (SQ)` <dbl> 97.7864496, 94.2051474, 110.6643855, NA, NA, …
$ `Cq Mean`                <dbl> 30.87718, 30.87718, 30.87718, 0.00000, 0.0000…
$ plate_id                 <chr> "B063809", "B063809", "B063809", "B063810", "…
$ Plate_number             <dbl> 12, 12, 12, 12, 12, 12, 12, 12, 12, 12, 12, 1…
$ gDNA_Plate_ID            <dbl> NA, NA, NA, NA, NA, NA, NA, NA, NA, NA, NA, N…
$ VMRC_Group               <chr> "VMRC_4", "VMRC_4", "VMRC_4", "VMRC_3", "VMRC…
$ Group_Fluor              <chr> "VMRC_4_HEX", "VMRC_4_HEX", "VMRC_4_HEX", "VM…
$ Target                   <chr> "122010", "122010", "122010", "185329", "1853…
$ gDNA_Plate_Well          <chr> NA, NA, NA, NA, NA, NA, NA, NA, NA, NA, NA, N…
$ VIBR_Sample_ID           <chr> "551", "551", "551", "551", "551", "551", "55…
$ InRedos                  <dbl> 1, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1, …
$ Dilution_Factor          <dbl> 5, 5, 5, 5, 5, 5, 5, 5, 5, 5, 5, 5, 5, 5, 5, …
$ Quant_Adjusted           <dbl> 488.932248, 471.025737, 553.321928, 0.000000,…
$ Copies_per_swab          <dbl> 122233.0620, 117756.4343, 138330.4819, 0.0000…

Merging 16S and LBP qPCR data

Since the two files have almost exactly the same columns, we merge them into a single file after some harmonization.

Note that there is one sample that has data for the LBP qPCR, but not for the 16S qPCR.

dplyr::full_join(
  qpcr_16S |> select(VIBR_Sample_ID) |> mutate(has_16S = TRUE) |> distinct(),
  qpcr_LBP |> select(VIBR_Sample_ID) |> mutate(has_LBP = TRUE) |> distinct()
) |>  dplyr::filter(is.na(has_16S) | is.na(has_LBP)) |> 
  dplyr::filter(!is.na(VIBR_Sample_ID), !(VIBR_Sample_ID %in% c("#N/A", "empty"))) |> 
  mutate(index = row_number()) |> 
  select(index, everything()) |> 
  gt(
    caption = "Clinical sample with data for LBP qPCR but not for 16S qPCR, or vice versa."
  )

Clinical sample with data for LBP qPCR but not for 16S qPCR, or vice versa.

index	VIBR_Sample_ID	has_16S	has_LBP
1	2171048	NA	TRUE

We also note that there is one sample on the same plate that has 6 replicates instead of 3 in the 16S qPCR.

qpcr_16S |> dplyr::filter(!is.na(VIBR_Sample_ID)) |> dplyr::count(VIBR_Sample_ID, gDNA_Plate_ID ) |> dplyr::filter(n > 3)

# A tibble: 1 × 3
  VIBR_Sample_ID gDNA_Plate_ID     n
  <chr>                  <dbl> <int>
1 2167612              2240890     6

qpcr_16S |> dplyr::filter(VIBR_Sample_ID == "2167612") |> select(Data_row, Well, Sample, Cq, Copies_per_swab)

# A tibble: 6 × 5
  Data_row Well  Sample    Cq Copies_per_swab
     <dbl> <chr> <chr>  <dbl>           <dbl>
1      163 I08   E4      22.4      218362435.
2      178 J07   E4      22.4      213446847.
3      179 J08   E4      22.4      222995668.
4      242 M10   G5      22.6      181004723.
5      258 N09   G5      22.5      195241505.
6      259 N10   G5      22.8      157625548.

As discussed with Michael, this is a mislabeling and these 6 are 3 replicates for 2 different samples. Since we have identified this issue already, we will fix it later in the code, using the extraction library plate number and position.

# We rename the standards in the 16S qPCR so they are numbered
std_16s <-
  qpcr_16S |> 
  dplyr::filter(str_detect(Content, "Std")) |> 
  select(Content, `Starting Quantity (SQ)`) |> 
  distinct() |> 
  arrange(`Starting Quantity (SQ)` |> desc()) |>
  mutate(std_nb = row_number())
  
qpcr_16S <- 
  qpcr_16S |> 
  dplyr::left_join(std_16s, by = join_by(Content, `Starting Quantity (SQ)`)) |> 
  mutate(
    Content = Content |> str_c("-", std_nb |> str_pad(width = 2, pad = "0")),
    Target = "16S"
        ) |> 
  select(-std_nb)

# We transform the VIBR_Sample_ID column to be consistent with the 16S qPCR
qpcr_LBP <- 
  qpcr_LBP |> 
  mutate(
    VIBR_Sample_ID = ifelse(VIBR_Sample_ID == "#N/A", NA_character_, VIBR_Sample_ID)
  )

# we merge the two datasets
qpcr <- 
  bind_rows(
    qpcr_LBP |> mutate(target_type = "LBP"), 
    qpcr_16S |> mutate(target_type = "16S rRNA")
    ) |> 
  relocate(target_type, .after = Target)

dictionary <- 
  tibble(original_name = colnames(qpcr)) |> 
  mutate(
    description = 
      case_when(
        (original_name == "Data_row") ~ 'Table row number',
        (original_name == "Well") ~ str_c("qPCR plate well ID. From ", min(qpcr$Well), " to ", max(qpcr$Well)),
        (original_name == "Fluor") ~ str_c('fluorescent dye used (one of ', qpcr$Fluor |> unique() |> str_c(collapse = ", "), ")"),
        (original_name == "Content") ~ 'Indicates whether well contains a sample, a standard, or a negative control.',
        (original_name == "Replicate") ~ 
          str_c(
            "The replicate number from ", min(qpcr$Replicate, na.rm = TRUE), " to ", max(qpcr$Replicate, na.rm = TRUE), 
            " (with ", sum(is.na(qpcr$Replicate)), " missing values). Values are repeated 495, or 990 times."
          ),
        (original_name == "Sample") ~ 'A qPCR plate-specific sample ID. This column wont be used or kept for downstream analyses.',
        (original_name == "Cq") ~ 'Quantification cycle (Cq) value.',
        (original_name == "Starting Quantity (SQ)") ~ 'Starting quantity (SQ) value; computed from the Cq values using the inverse calibration function (calibration curve fitted using the expected SQ values for the standards).',
        (original_name == "Cq Mean") ~ 'Mean across replicates',
        (original_name == "plate_id") ~ str_c('qPCR plate ID; there are ', qpcr$plate_id |> unique() |> length()," unique plate IDs in total."),
        (original_name == "Plate_number") ~ str_c("The extraction plate number; there are ", qpcr$Plate_number |> unique() |> length()," unique plate numbers in total (5 identical `plate_id` per `Plate_number`)."),
        (original_name == "gDNA_Plate_ID") ~ 'The extraction plate ID',
        (original_name == "VMRC_Group") ~ str_c('The group of the LBP strains. There are ', qpcr$VMRC_Group |> unique() |> na.omit() |> length()," unique VMRC groups in total (", qpcr$VMRC_Group |> unique() |> na.omit() |> sort() |> str_c(collapse = ", "), ")"),
        (original_name == "Group_Fluor") ~ 'Concatenation of the `VMRC_Group` and `Fluor` columns',
        (original_name == "Target") ~ str_c('The target "gene" (here taxa/strain). There are ', qpcr$Target |> unique() |> length(), " unique targets in total (", qpcr$Target |> unique() |> str_c(collapse = ", "),")"),
        (original_name == "target_type") ~ "Whether the target is a LBP strain or the 16S rRNA gene target",
        (original_name == "gDNA_Plate_Well") ~ str_c('The concatenation of the gDNA plate ID and the plate well; there are ', qpcr$gDNA_Plate_Well |> unique() |> length()," unique gDNA plate wells in total in the format `[gDNA_Plate_ID]_[Well]` (e.g., ", qpcr$gDNA_Plate_Well |> unique() |> extract(10),")"),
        (original_name == "VIBR_Sample_ID") ~ str_c("VIBRANT sample ID; there are ", qpcr$VIBR_Sample_ID |> unique() |> length(), " unique VIBRANT sample IDs in total)"),
        (original_name == "Dilution_Factor") ~ str_c('The qPCR plate specific dilution factor. There are ', qpcr$Dilution_Factor |> unique() |> length()," unique dilution factors in total (", qpcr$Dilution_Factor |> unique()  |> sort() |> str_c(collapse = ", "),")"),
        (original_name == "Quant_Adjusted") ~ str_c("Quantification value adjusted by the dilution factor; range from ", min(qpcr$Quant_Adjusted, na.rm = TRUE)," to ", max(qpcr$Quant_Adjusted, na.rm = TRUE)),
        (original_name == "Copies_per_swab") ~ str_c("The number of target copies per swab, computed from the adjusted quantification values. Ranges from ", min(qpcr$Copies_per_swab, na.rm = TRUE)," to ", max(qpcr$Copies_per_swab, na.rm = TRUE)),
         (original_name == "InRedos") ~ str_c("Whether the sample was part of the samples that were redone (= re-extracted)."),
        TRUE ~ "????"
      )
  )

# dictionary |> gt()

Tidy names

We tidy the column names for consistency and readability.

qpcr <- qpcr |> janitor::clean_names()
qpcr <- qpcr |> 
  dplyr::rename(
    strain_group_qpcr = vmrc_group,
    starting_quantity = starting_quantity_sq,
    pcr_plate_id = plate_id,
    ext_lib_plate_nb = plate_number,
    ext_lib_plate_id = g_dna_plate_id,
    ext_lib_position = g_dna_plate_well
  )

dictionary <- 
  dictionary |> 
  mutate(name = colnames(qpcr)) |> 
  select(name, everything())

The new column names are:

dictionary |> 
  gt() |> 
  cols_label(
    name = "New column name",
    original_name = "Original column name",
    description = "Description"
  ) 

New column name	Original column name	Description
data_row	Data_row	Table row number
well	Well	qPCR plate well ID. From A01 to P24
fluor	Fluor	fluorescent dye used (one of HEX, Cy5, FAM, SYBR)
content	Content	Indicates whether well contains a sample, a standard, or a negative control.
replicate	Replicate	The replicate number from 1 to 96 (with 4193 missing values). Values are repeated 495, or 990 times.
sample	Sample	A qPCR plate-specific sample ID. This column wont be used or kept for downstream analyses.
cq	Cq	Quantification cycle (Cq) value.
starting_quantity	Starting Quantity (SQ)	Starting quantity (SQ) value; computed from the Cq values using the inverse calibration function (calibration curve fitted using the expected SQ values for the standards).
cq_mean	Cq Mean	Mean across replicates
pcr_plate_id	plate_id	qPCR plate ID; there are 72 unique plate IDs in total.
ext_lib_plate_nb	Plate_number	The extraction plate number; there are 12 unique plate numbers in total (5 identical `plate_id` per `Plate_number`).
ext_lib_plate_id	gDNA_Plate_ID	The extraction plate ID
strain_group_qpcr	VMRC_Group	The group of the LBP strains. There are 5 unique VMRC groups in total (VMRC_1, VMRC_2, VMRC_3, VMRC_4, VMRC_5)
group_fluor	Group_Fluor	Concatenation of the `VMRC_Group` and `Fluor` columns
target	Target	The target "gene" (here taxa/strain). There are 16 unique targets in total (122010, 185329, C0006A1, C0022A1, C0028A1, C0059E1, C0112A1, C0175A1, FF00004, FF00018, FF00051, FF00064, FF00072, UC101, UC119, 16S)
target_type	target_type	Whether the target is a LBP strain or the 16S rRNA gene target
ext_lib_position	gDNA_Plate_Well	The concatenation of the gDNA plate ID and the plate well; there are 1221 unique gDNA plate wells in total in the format `[gDNA_Plate_ID]_[Well]` (e.g., 2240900_G2)
vibr_sample_id	VIBR_Sample_ID	VIBRANT sample ID; there are 1031 unique VIBRANT sample IDs in total)
in_redos	InRedos	Whether the sample was part of the samples that were redone (= re-extracted).
dilution_factor	Dilution_Factor	The qPCR plate specific dilution factor. There are 7 unique dilution factors in total (5, 10, 20, 30, 33.333, 133.333)
quant_adjusted	Quant_Adjusted	Quantification value adjusted by the dilution factor; range from 0 to 3e+08
copies_per_swab	Copies_per_swab	The number of target copies per swab, computed from the adjusted quantification values. Ranges from 0 to 7.5e+10

qpcr <- 
  qpcr |> 
  mutate(
    in_redos = (in_redos == 1) |> replace_na(FALSE) # Convert to logical
  )

Sample types and unique sample IDs

We define a qpcr_sample_type variable to summarize information from vibr_sample_id and sample columns.

qpcr <-  
  qpcr |> 
  mutate(
    qpcr_sample_type = 
      case_when(
        is.na(vibr_sample_id) & str_detect(content, "Std") ~ "Standard",
        is.na(vibr_sample_id) & str_detect(sample, "water") ~ "Water",
        str_detect(vibr_sample_id, "EQ") ~ "Control sample",
        str_detect(vibr_sample_id, "empty") ~ "Empty",
        is.na(vibr_sample_id) ~ "Water or empty",
        TRUE ~ "VIBRANT clinical sample"
      )
  )

# qpcr |> dplyr::filter(target == "16S") |> arrange(qpcr_sample_type) |> View()

qpcr |> dplyr::count(qpcr_sample_type, name = "n_wells") |> arrange(-n_wells) |> gt()

qpcr_sample_type	n_wells
VIBRANT clinical sample	49727
Standard	4068
Control sample	2112
Empty	1890
Water	540
Water or empty	126

tmp <- 
  qpcr |> 
  dplyr::filter(qpcr_sample_type == "VIBRANT clinical sample", target == target[1]) |>
  group_by(vibr_sample_id) |> 
  summarize(
    n_replicates = n(),
    n_plates = ext_lib_plate_nb |> unique() |> length(),
    any_in_redos = any(in_redos)
  )

# tmp |> dplyr::count(n_plates, any_in_redos) # All samples's replicates are on a single plate

# tmp |> dplyr::count(n_replicates, n_plates, any_in_redos) # All samples have 3 replicates

We note that almost all samples’s replicates are on a single extraction plate (ext_lib_plate_nb or ext_lib_plate_id); and almost all samples have the same number of replicates (3). A few samples (those in_redos) are on two plates and have 6 replicates.

tmp <- 
  qpcr |> 
  dplyr::filter(qpcr_sample_type == "VIBRANT clinical sample", target == "16S") |>
  group_by(vibr_sample_id) |> 
  summarize(
    n_replicates = n(),
    n_plates = ext_lib_plate_nb |> unique() |> length()
  )

# tmp |> dplyr::count(n_plates) # All samples's replicates are on a single plate

# tmp |> dplyr::count(n_replicates) # All samples have 3 replicates

tmp |> 
  dplyr::filter(n_replicates == 6) |> 
  dplyr::left_join(qpcr |> dplyr::filter(target == "16S"), by = join_by(vibr_sample_id)) |> 
  select(vibr_sample_id, ext_lib_plate_nb, ext_lib_position, pcr_plate_id, data_row, well, cq) |> 
  gt()

We’ll fix that below.

qpcr <- 
  qpcr |> 
  mutate(
    well_col = well |> str_sub(1, 1), 
    well_row = well |> str_sub(2,3) |> as.integer()
    ) |> 
  relocate(well_col, well_row, .after = well)

We also define a unique “qPCR sample ID” according to each sample type.

qpcr <- 
  qpcr |> 
  mutate(
    qpcr_sample_id = 
      case_when(
        (qpcr_sample_type == "VIBRANT clinical sample") ~ vibr_sample_id,
        (qpcr_sample_type == "Control sample") ~ vibr_sample_id,
        (qpcr_sample_type == "Empty") ~ 
          str_c(vibr_sample_id,"_plate_", 
                ext_lib_plate_nb |> str_pad(width = 2, pad = 0),"_", sample),
        (qpcr_sample_type == "Water") ~ 
          str_c("water_plate", 
                ext_lib_plate_nb |> str_pad(width = 2, pad = 0)),
        (qpcr_sample_type == "Water or empty") ~ 
          str_c("water_or_empty_plate", 
                ext_lib_plate_nb |> str_pad(width = 2, pad = 0),"_", sample),
        (qpcr_sample_type == "Standard") ~ 
          str_c("std_", content |> str_remove("Std-"), 
                "_plate_", ext_lib_plate_nb |> str_pad(width = 2, pad = 0)),
        TRUE ~ NA_character_
      )
  ) |> 
  group_by(qpcr_sample_id, target) |>
  arrange(ext_lib_plate_nb, well_col, well_row) |>
  mutate(replicate_nb = row_number()) |> 
  ungroup() |> 
  mutate(qpcr_uid = str_c(qpcr_sample_id, "_r", replicate_nb))

# qpcr |> dplyr::filter(replicate_nb > 3) |> View()

For the LBP strains, the plate layout is as follows for the first 11 extraction plates:

qpcr_sample_type_colors <- c(
  "VIBRANT clinical sample" = "dodgerblue2",
  "Control sample" = "turquoise",
  "Standard" = "gold",
  "Water" = "lightblue1",
  "Empty" = "gray",
  "Water or empty" = "gray80"
)

qpcr |> 
  dplyr::filter(target != "16S", ext_lib_plate_nb <= 11) |> 
  select(ext_lib_plate_nb, well_col, well_row, qpcr_sample_type, sample) |>
  distinct() |>
  ggplot() +
  aes(x =  well_row |> factor(), y = well_col |> factor() |> fct_rev(), fill = qpcr_sample_type) +
  facet_wrap(~ ext_lib_plate_nb) +
  geom_tile() +
  geom_path(aes(group = sample), col = "black", alpha = 0.5) +
  geom_point() +
  scale_fill_manual(values = qpcr_sample_type_colors) +
  xlab("Well column") + ylab("Well row") +
  labs(caption = "Black lines connect replicates") 

For the 12th plate, the layout is a little bit different and the first PCR plate (for the first group of 3 target) is different from the 4 other:

qpcr |> 
  dplyr::filter(target != "16S", ext_lib_plate_nb == 12) |> 
  select(ext_lib_plate_nb, pcr_plate_id, well_col, well_row, qpcr_sample_type, sample, strain_group_qpcr) |>
  distinct() |>
  ggplot() +
  aes(x =  well_row |> factor(), y = well_col |> factor() |> fct_rev(), fill = qpcr_sample_type) +
  facet_wrap(~ pcr_plate_id + strain_group_qpcr) +
  geom_tile() +
  geom_path(aes(group = sample), col = "black", alpha = 0.5) +
  geom_point() +
  scale_fill_manual(values = qpcr_sample_type_colors) +
  xlab("Well column") + ylab("Well row") +
  labs(caption = "Black lines connect replicates") 

For the 16S plates, the layout is a little different:

qpcr |> 
  dplyr::filter(target == "16S") |> 
  select(ext_lib_plate_nb, pcr_plate_id, well_col, well_row, qpcr_sample_type, qpcr_sample_id) |>
  distinct() |>
  ggplot() +
  aes(x =  well_row |> factor(), y = well_col |> factor() |> fct_rev(), fill = qpcr_sample_type, col = qpcr_sample_type) +
  facet_wrap(~ ext_lib_plate_nb + pcr_plate_id) +
  geom_tile(alpha = 0.25) +
  geom_path(aes(group = qpcr_sample_id), alpha = 0.5, linewidth = 2) +
  geom_point() +
  scale_fill_manual(values = qpcr_sample_type_colors) +
  scale_color_manual(values = qpcr_sample_type_colors) +
  xlab("Well column") + ylab("Well row") +
  labs(caption = "Lines connect replicates") 

Manifest data

We merge the qPCR data with the metagenomics manifest data to add the participant ID (pid) and (visit_code).

But before we do that, we fix the qpcr_sample_id for that sample that had 6 replicates instead of 3 in the 16S qPCR because, after discussion with Michael, we know that one of these barcode was wrong.

six_replicates <- 
  qpcr |> 
  dplyr::filter(target == "16S") |> 
  group_by(qpcr_sample_id) |> 
  mutate(n_replicates = n(), none_on_plate_12 = !any(ext_lib_plate_nb == 12)) |> 
  ungroup() |> 
  dplyr::filter(n_replicates > 3, none_on_plate_12) |> 
  select(target, vibr_sample_id, qpcr_sample_id, qpcr_uid, ext_lib_position, ext_lib_plate_nb, ext_lib_plate_id, n_replicates)

six_replicates |> gt()

target	vibr_sample_id	qpcr_sample_id	qpcr_uid	ext_lib_position	ext_lib_plate_nb	ext_lib_plate_id	n_replicates
16S	2167612	2167612	2167612_r1	2240890_E4	1	2240890	6
16S	2167612	2167612	2167612_r2	2240890_E4	1	2240890	6
16S	2167612	2167612	2167612_r3	2240890_E4	1	2240890	6
16S	2167612	2167612	2167612_r4	2240890_G5	1	2240890	6
16S	2167612	2167612	2167612_r5	2240890_G5	1	2240890	6
16S	2167612	2167612	2167612_r6	2240890_G5	1	2240890	6

We see that these 6 replicates have 2 different positions on the extraction library plate so we will be able to fix their sample ID from using extraction plate data from the technical metadata manifest using the concatenation of ext_lib_plate_id and ext_lib_position.

technical_metadata_file <- 
  list.files(
    path = get_01_output_dir(), 
    pattern = "02_technical_metadata_agg_.*\\.rds$", full.names = TRUE
  ) |> 
  sort(decreasing = TRUE) |>
  extract(1)

technical_metadata <- readRDS(technical_metadata_file)

rm(technical_metadata_file)

# SE_mg <- 
#   readRDS(
#     list.files(
#       path = get_output_dir(data_source = data_source), 
#       pattern = "02_se_mg_.*\\.rds$", full.names = TRUE
#     ) |> 
#       sort(decreasing = TRUE) |>
#       extract(1) 
#   )

six_replicates |> 
  mutate(ext_lib_well = ext_lib_position |> str_remove("[0-9]*_")) |> 
  dplyr::left_join(
    technical_metadata |> 
      select(swab_barcode, ext_lib_plate_nb, ext_lib_plate_id, ext_lib_position) |>
      dplyr::rename(
        ext_lib_plate_id_mg_manifest = ext_lib_plate_id,
        ext_lib_well = ext_lib_position
      )
  ) |> gt()

target	vibr_sample_id	qpcr_sample_id	qpcr_uid	ext_lib_position	ext_lib_plate_nb	ext_lib_plate_id	n_replicates	ext_lib_well	swab_barcode	ext_lib_plate_id_mg_manifest
16S	2167612	2167612	2167612_r1	2240890_E4	1	2240890	6	E4	2171048	2316297
16S	2167612	2167612	2167612_r2	2240890_E4	1	2240890	6	E4	2171048	2316297
16S	2167612	2167612	2167612_r3	2240890_E4	1	2240890	6	E4	2171048	2316297
16S	2167612	2167612	2167612_r4	2240890_G5	1	2240890	6	G5	2167612	2316297
16S	2167612	2167612	2167612_r5	2240890_G5	1	2240890	6	G5	2167612	2316297
16S	2167612	2167612	2167612_r6	2240890_G5	1	2240890	6	G5	2167612	2316297

One thing that is weird is that the ext_lib_plate_id in the technical metadata manifest is not the same as the one in the qPCR data:

qpcr |> 
  dplyr::count(ext_lib_plate_nb, ext_lib_plate_id) |> 
  gt(caption = "Extraction library plate nb and IDs as found in the qPCR data.")

Extraction library plate nb and IDs as found in the qPCR data.

ext_lib_plate_nb	ext_lib_plate_id	n
1	2240890	4992
2	2240900	4992
3	2316397	4992
4	2316390	4992
5	2316420	4992
6	2316419	4992
7	2316095	4992
8	2316093	4992
9	2315117	4992
10	2316391	4992
11	2329564	4991
12	45786	45
12	2334591	153
12	NA	3354

technical_metadata |> 
  dplyr::count(ext_lib_plate_nb, ext_lib_plate_id) |> 
  gt(caption = "Extraction library plate nb and IDs as found in the metagenomics technical metadata.")

Extraction library plate nb and IDs as found in the metagenomics technical metadata.

ext_lib_plate_nb	ext_lib_plate_id	n
1	2316297	89
2	2240891	88
3	2316307	86
4	2316306	88
5	2316310	87
6	2316311	89
7	2316094	88
8	2316097	88
9	2316392	79
10	2316393	82
11	2329564	85
12	2240891	1
12	2316094	2
12	2316097	2
12	2316297	2
12	2316306	1
12	2316307	2
12	2316310	2
12	2316311	1
12	2316392	2
12	2316393	2
NA	NA	63

Warning

@Michael: What are your thoughts on this? Am I assuming they should be the same when there is no reason for them to be the same?

Regardless, since the ext_lib_plate_nb seems to match, we’ll use this to fix that 6-replicates sample. From the table above, it looks like the qpcr_sample_id for the 16S qPCR located in well E4 should be 2171048 instead of 2167612.

We fix their vibr_sample_id, qpcr_sample_id, qpcr_uid, and replicate_nb columns.

qpcr <- 
  qpcr |> 
  mutate(
    needs_change = 
      (qpcr_sample_id == "2167612" & 
         target == "16S" & 
         ext_lib_plate_nb == 1 & 
         ext_lib_position == "2240890_E4"),
    needs_replicates_change = 
      (qpcr_sample_id == "2167612" & 
         target == "16S" & 
         ext_lib_plate_nb == 1 & 
         ext_lib_position == "2240890_G5"),
    qpcr_sample_id = case_when(
      needs_change ~ "2171048",
      TRUE ~ qpcr_sample_id
    ),
    vibr_sample_id = case_when(
      needs_change ~ "2171048",
      TRUE ~ vibr_sample_id
    ),
    qpcr_uid = case_when(
      needs_change ~ qpcr_uid |> str_replace("2167612", "2171048"),
      needs_replicates_change ~ qpcr_uid |> str_replace("r4", "r1") |> str_replace("r5", "r2") |> str_replace("r6", "r3"),
      TRUE ~ qpcr_uid
    ),
    replicate_nb = 
      case_when(
        needs_replicates_change ~ replicate_nb - 3L,
        TRUE ~ replicate_nb
      )
  ) |> 
  select(-needs_change, needs_replicates_change)

Now that this is fixed, we can merge the technical metadata with the qPCR data to be able to build their uid (VIBRANT cross-assay unique identifier) and identify their sample_type, control_type, etc.

qpcr <- 
  qpcr |> 
  dplyr::left_join(
    technical_metadata |> 
      select(
        swab_barcode,
        uid, mg_uid, 
        mg_pid, mg_visit_code, 
        sample_type, control_type
      ),
    by = join_by("qpcr_sample_id" == "swab_barcode")
  )

qpcr |>
  dplyr::filter(is.na(mg_uid)) |> 
  dplyr::count(content, vibr_sample_id) |> 
  View()

samples_without_entries_in_mg_manifest <- 
  qpcr |> 
  dplyr::filter(qpcr_sample_type %in% c("VIBRANT clinical sample", "Control sample")) |>
  dplyr::filter(is.na(uid)) |> 
  select(qpcr_sample_id, qpcr_sample_type) |> 
  distinct()  |> 
  arrange(qpcr_sample_type, qpcr_sample_id) 

There are 1 clinical or vibrant positive/negative control samples without a matching entry in the metagenomics manifest.

samples_without_entries_in_mg_manifest |> 
  # dplyr::count(qpcr_sample_type) |>
  gt()

qpcr_sample_id	qpcr_sample_type
2320838	VIBRANT clinical sample

In an email from June 16th 2025, Michael indicated that this is a negative biological sample (Unused swab + C2).

We fix it manually for data completion:

qpcr <- 
  qpcr |> 
  mutate(
    sample_type = 
      case_when(
        qpcr_sample_id == "2320838" ~ "Negative control",
        TRUE ~ sample_type |> as.character()
      ) |> factor(levels = levels(qpcr$sample_type)),
    control_type =
      case_when(
        qpcr_sample_id == "2320838" ~ "Unused swab + C2",
        TRUE ~ control_type |> as.character()
      ) |> factor(levels = levels(qpcr$control_type)),
    qpcr_sample_type = 
      case_when(
        qpcr_sample_id == "2320838" ~ "Control sample",
        TRUE ~ qpcr_sample_type
      )
  )

For these samples, the current uid is missing. We change it to the qpcr_sample_id when NA.

qpcr <- 
  qpcr |> 
  mutate(
    uid = case_when(
      is.na(uid) & 
        (qpcr_sample_type %in% c("VIBRANT clinical sample", "Control sample")) ~ 
        qpcr_sample_id,
      TRUE ~ uid
    )
  )

mg_samples <- 
  technical_metadata |> 
  select(
    swab_barcode,
    uid, mg_uid, 
    mg_pid,mg_visit_code, 
    sample_type, control_type,
    ext_lib_plate_nb, no_mg_data
  ) |> 
  distinct() |> 
  dplyr::left_join(
    qpcr |> 
      select(qpcr_sample_id, qpcr_sample_type) |> 
      distinct() |> 
      dplyr::filter(qpcr_sample_type %in% c("VIBRANT clinical sample", "Control sample")),
    by = join_by("swab_barcode" == "qpcr_sample_id")
  ) |> 
  arrange(uid)

# mg_samples |> dplyr::filter(is.na(qpcr_sample_type)) |> nrow()

There are 1 samples for which we have metagenomics data but no qPCR data:

mg_samples |> dplyr::filter(is.na(qpcr_sample_type)) |> gt()

swab_barcode	uid	mg_uid	mg_pid	mg_visit_code	sample_type	control_type	ext_lib_plate_nb	no_mg_data	qpcr_sample_type
MC1	MC1	PC_MC1	Mock1	NA	Positive control	Mock 1	NA	FALSE	NA

mg_samples <- 
  technical_metadata |> 
  select(
    swab_barcode,
    uid, mg_uid, 
    mg_pid,mg_visit_code, 
    sample_type, control_type,
    ext_lib_plate_nb, no_mg_data
  ) |> 
  distinct() |> 
  dplyr::left_join(
    qpcr |>
      select(qpcr_sample_id, qpcr_sample_type, target) |> 
      distinct() |> 
      dplyr::filter(
        target == "16S",
        qpcr_sample_type %in% c("VIBRANT clinical sample", "Control sample")
        ),
    by = join_by("swab_barcode" == "qpcr_sample_id")
  ) |> 
  arrange(uid)

And there are 1 samples for which we have metagenomics data but no 16S qPCR data:

mg_samples |> dplyr::filter(is.na(qpcr_sample_type)) |> gt()

swab_barcode	uid	mg_uid	mg_pid	mg_visit_code	sample_type	control_type	ext_lib_plate_nb	no_mg_data	qpcr_sample_type	target
MC1	MC1	PC_MC1	Mock1	NA	Positive control	Mock 1	NA	FALSE	NA	NA

rm(mg_samples, samples_without_entries_in_mg_manifest, tmp)

qpcr |> 
  dplyr::count(qpcr_sample_type, sample_type, control_type)

qpcr <- 
  qpcr |> 
  mutate(
    sample_type = 
      case_when(
        is.na(sample_type) & (qpcr_sample_type == "VIBRANT clinical sample") ~ "Clinical sample",
        is.na(sample_type) & (qpcr_sample_type %in% c("Empty", "Water", "Water or empty")) ~ "Technical negative control",
        is.na(sample_type) & (qpcr_sample_type == "Standard") ~ "Standard",
        TRUE ~ sample_type
      ),
    control_type =
      case_when(
        is.na(control_type) & (qpcr_sample_type != "VIBRANT clinical sample") ~ qpcr_sample_type,
        is.na(control_type) & (qpcr_sample_type == "VIBRANT clinical sample") ~ "",
        TRUE ~ control_type
      )
  )

Target’s plate layout and fluorescent probes

The plate x Fluorescence x Target layout is as follows:

qpcr |> 
  dplyr::count(fluor, target, strain_group_qpcr, pcr_plate_id, ext_lib_plate_nb) |> 
  arrange(ext_lib_plate_nb, strain_group_qpcr, pcr_plate_id, fluor) |> 
  mutate(
    target = target |> fct_inorder(), 
    plate_nb = str_c("ext. plate: ", ext_lib_plate_nb) |> fct_inorder(),
    pcr_plate_id = pcr_plate_id |> fct_inorder()
    ) |> 
  ggplot() +
  aes(
    x = pcr_plate_id,
    y = target |> factor() |> fct_rev(),
    fill = fluor
  ) +
  geom_tile() +
  facet_grid(strain_group_qpcr ~ plate_nb, scales = "free", space = "free") +
  xlab("qPCR plate ID") +
  ylab("Target") +
  scale_fill_manual(
    name = "Fluor.",
    values = c("FAM" = "dodgerblue1", "HEX" = "green3", "Cy5" = "#F8766D", "SYBR" = "green")
  ) +
  theme(
    legend.position = "bottom",
    axis.text.x = element_text(angle = 45, hjust = 1),
    strip.text.y = element_text(angle = 0)
  )

Potential batch effects due to extraction should be checked for ext_lib_plate_id and those due to the qPCR itself using the pcr_plate_id.

LBP strain information

We also add the LBP strain information to the qPCR data.

SE_mg <-
  readRDS(
    list.files(
      path = get_01_output_dir(),
      pattern = "02_se_mg_.*\\.rds$", full.names = TRUE
    ) |>
      sort(decreasing = TRUE) |>
      extract(1)
  )

qpcr <- 
  qpcr |> 
  dplyr::left_join(
    SE_mg |>
      rowData() |> as.data.frame() |> as_tibble() |> 
      dplyr::filter(!is.na(LBP)) |> 
      select(taxon_id, taxon_label, LBP, strain_id, strain_origin, biose_id) |> 
      dplyr::rename(target = taxon_id),
    by = join_by(target)
  )

qpcr <- 
  qpcr |> 
  mutate(taxon_label = taxon_label |> str_replace_na("16S rRNA gene target"))

rm(SE_mg)

qpcr |> 
  arrange(strain_group_qpcr, strain_origin) |>
  mutate(target = target |> fct_inorder()) |> 
  ggplot() +
  aes(x = target, y = strain_group_qpcr, col = strain_group_qpcr) +
  geom_point() +
  facet_grid(. ~ LBP + strain_origin, scales = "free", space = "free")  +
  xlab("Target") + ylab("VMRC group") +
  guides(col = "none") +
  theme(
    axis.text.x = element_text(angle = 45, hjust = 1)
  ) 

Relationships between columns

Observations and calibration curves

Observed values are the cq values.

starting_quantity are computed from the cq values using the calibration curves fitted on the standard values.

\[C = f(SQ^*)\]

where \(C\) is the observed cq value for the standard, \(SQ^*\) is the known starting quantity starting_quantity for the standards, and \(f\) is the calibration function.

Note

I assume that, either the standards are not diluted, and that the quant_adjusted and copies_per_swabs are not correct for the standards, or that the standards are diluted and that the calibration curves are fitted on the quant_adjusted:

qpcr |>
  dplyr::filter(qpcr_sample_type == "Standard", target != "16S") |> 
  select(
    sample, starting_quantity, dilution_factor, quant_adjusted, copies_per_swab
  ) |> 
  distinct() |> 
  arrange(sample, dilution_factor) |> 
  gt()

sample	starting_quantity	dilution_factor	quant_adjusted	copies_per_swab
std 10^1	1e+01	5	5e+01	1.25e+04
std 10^1	1e+01	10	1e+02	2.50e+04
std 10^1	1e+01	20	2e+02	5.00e+04
std 10^1	1e+01	30	3e+02	7.50e+04
std 10^1	1e+01	NA	NA	NA
std 10^2	1e+02	5	5e+02	1.25e+05
std 10^2	1e+02	10	1e+03	2.50e+05
std 10^2	1e+02	20	2e+03	5.00e+05
std 10^2	1e+02	30	3e+03	7.50e+05
std 10^2	1e+02	NA	NA	NA
std 10^3	1e+03	5	5e+03	1.25e+06
std 10^3	1e+03	10	1e+04	2.50e+06
std 10^3	1e+03	20	2e+04	5.00e+06
std 10^3	1e+03	30	3e+04	7.50e+06
std 10^3	1e+03	NA	NA	NA
std 10^4	1e+04	5	5e+04	1.25e+07
std 10^4	1e+04	10	1e+05	2.50e+07
std 10^4	1e+04	20	2e+05	5.00e+07
std 10^4	1e+04	30	3e+05	7.50e+07
std 10^4	1e+04	NA	NA	NA
std 10^5	1e+05	5	5e+05	1.25e+08
std 10^5	1e+05	10	1e+06	2.50e+08
std 10^5	1e+05	20	2e+06	5.00e+08
std 10^5	1e+05	30	3e+06	7.50e+08
std 10^5	1e+05	NA	NA	NA
std 10^6	1e+06	5	5e+06	1.25e+09
std 10^6	1e+06	10	1e+07	2.50e+09
std 10^6	1e+06	20	2e+07	5.00e+09
std 10^6	1e+06	30	3e+07	7.50e+09
std 10^6	1e+06	NA	NA	NA
std 10^7	1e+07	5	5e+07	1.25e+10
std 10^7	1e+07	10	1e+08	2.50e+10
std 10^7	1e+07	20	2e+08	5.00e+10
std 10^7	1e+07	30	3e+08	7.50e+10
std 10^7	1e+07	NA	NA	NA

qpcr |>
  dplyr::filter(qpcr_sample_type == "Standard") |> 
  arrange(-dilution_factor) |>
  ggplot() +
  aes(
    # x = starting_quantity |> log10(), 
    x = quant_adjusted |> log10(),
    y = cq, 
    col = dilution_factor |> factor()
  ) +
  geom_point(size = 1, alpha = 0.2) +
  scale_color_manual(
    "Dilution factor", 
    values = colorRampPalette(c("black", "dodgerblue1"))(4)
    ) +
  facet_wrap(. ~ strain_group_qpcr + target, ncol = 9) 

For all clinical samples, the starting_quantity is computed from the cq values using the calibration curve fitted on the standard values.

\[\hat{SQ} = f^{-1}(C)\]

where \(\hat{SQ}\) is the estimated starting quantity starting_quantity for the clinical samples, \(C\) is the observed cq value for the clinical sample, and \(f^{-1}\) is the inverse of the calibration function.

If \(C\) is not observed (i.e., assumed to be larger than the total number of cycles), then \(\hat{SQ}\) is estimated as 0.

# qpcr |> 
#   ggplot() +
#   aes(x = starting_quantity |> log10(), y = cq, col = qpcr_sample_type) +
#   geom_point(size = 0.5, alpha = 0.4) +
#   facet_grid(strain_group_qpcr + target ~ ext_lib_plate_nb) 


qpcr |> 
  ggplot() +
  aes(
    x = starting_quantity |> log10(), 
    y = cq, 
    col = ext_lib_plate_nb |> factor(),
    size = (qpcr_sample_type == "Standard")
    ) +
  geom_point(alpha = 0.5) +
  ggh4x::facet_nested_wrap(vars(strain_group_qpcr, target), ncol = 6) +
  scale_size_manual("Standard", values = c(0.2, 1)) +
  scale_color_viridis_d("Extraction plate number", direction = -1, option = "A", end = 0.8) 

It looks like \(f\) is a linear function of \(\log(SQ^*)\): \(C = \beta_0 + \beta_1 \log(SQ^*)\). This seems appropriate for the LBP strains, but for the total 16S, the standard curves are not linear. Probably not too much of an issue and unlikely to create huge batch effects.

Adjusted quantities

Adjusted quantities (quant_adjusted) are then computed from the estimated starting_quantity values using the dilution factors.

\(\hat{Q} = \hat{SQ} \times d\)

where \(\hat{Q}\) is the estimated adjusted quantity (quant_adjusted), \(\hat{SQ}\) is the estimated starting quantity (starting_quantity) for the clinical samples, and \(d\) is the dilution factor.

qpcr |>
  ggplot() +
  aes(
    x = starting_quantity |> log10(), 
    y = quant_adjusted |> log10(), 
    col = dilution_factor |> factor()
    ) +
  geom_point() +
  ggh4x::facet_nested_wrap(vars(strain_group_qpcr, target), ncol = 9) +
  scale_color_manual(
    "Dilution factor", 
    values = colorRampPalette(c("dodgerblue1", "gray90"))(6)
    )

quant_adjusted = starting_quantity x dilution_factor:

qpcr |>
  ggplot() +
  aes(
    x = (starting_quantity * dilution_factor) |> log10(), 
    y = (quant_adjusted) |> log10(), 
    col = dilution_factor |> factor()
    ) +
  geom_point() +
  ggh4x::facet_nested_wrap(vars(strain_group_qpcr, target), ncol = 9) +
  scale_color_manual(
    "Dilution factor", 
    values = colorRampPalette(c("dodgerblue1", "gray90"))(6)
    )

Copies per swab

copies_per_swab are the adjusted_quant multiplied by a constant factor estimated to be the number of bacteria in 1 concentration unit.

Here, that number is 250 for all LBP targets.

# strain_group_qpcr + target

qpcr |>
  dplyr::filter(target != "16S") |> 
  ggplot() +
  aes(
    x = (quant_adjusted * 250) |> log10(), 
    y = copies_per_swab |> log10(), 
    col =  target
    ) +
  geom_abline(intercept = 0, slope = 1, col = "gray") +
  geom_point(size = 0.5, alpha = 0.4) +
  facet_wrap(. ~ ext_lib_plate_nb , ncol = 6) 

For the 16S target, that number is 500, except for plate 12, where it is 250.

qpcr |>
  dplyr::filter(target == "16S") |> 
  mutate(
    r = copies_per_swab / (quant_adjusted )
  ) |> 
  pull(r) |> 
  hist()

This was actually a mistake and it should be 250 for all plates for the 16S target.

We fix this here:

qpcr <- 
  qpcr |> 
  mutate(
    copies_per_swab = 
      case_when(
        (qpcr_sample_type != "Standard") & 
          (target == "16S") & 
          (ext_lib_plate_nb < 12) ~ 
          copies_per_swab / 2,
        TRUE ~ 
          copies_per_swab
      )
  )

Dilution factors

Dilution factors are defined per qPCR plate (and consequently, are the same for the 3 targets in the same VMRC group)

qpcr |> 
  dplyr::count(target, dilution_factor) 

qpcr |> 
  group_by(pcr_plate_id, target) |>
  summarize(n_dilution_factors = dilution_factor |> unique() |> length()) 

#|

qpcr |> 
  select(
    starts_with("well"), 
    pcr_plate_id, 
    strain_group_qpcr, 
    ext_lib_plate_nb, 
    dilution_factor
    ) |> 
  distinct() |> 
  ggplot() +
  aes(
    x = well_row |> factor(), 
    y = well_col |> fct_rev(), 
    fill = dilution_factor |> factor()
    ) +
  geom_tile() +
  facet_grid(strain_group_qpcr ~ ext_lib_plate_nb, scales = "free", space = "free") +
  scale_fill_manual(
    "Dilution factor", 
    values = colorRampPalette(c("dodgerblue1", "gray90"))(6)
    ) +
  xlab("Well column") + ylab("Well row") 

Exploratory & QC analyses

CQ and Copies per swabs per well

qpcr |> 
  select(
    starts_with("well"), 
    pcr_plate_id, 
    target,
    strain_group_qpcr, 
    ext_lib_plate_nb, 
    cq
    ) |> 
  distinct() |> 
  ggplot() +
  aes(
    x = well_row |> factor(), 
    y = well_col |> fct_rev(), 
    fill = cq
    ) +
  geom_tile() +
  ggh4x::facet_nested(cols = vars(ext_lib_plate_nb), rows = vars(strain_group_qpcr, target), scales = "free", space = "free") +
  xlab("Well column") + ylab("Well row") 

qpcr |> 
  select(
    starts_with("well"), 
    pcr_plate_id, 
    target,
    strain_group_qpcr, 
    ext_lib_plate_nb, 
    copies_per_swab
    ) |> 
  distinct() |> 
  ggplot() +
  aes(
    x = well_row |> factor(), 
    y = well_col |> fct_rev(), 
    fill = copies_per_swab |> asinh()
    ) +
  geom_tile() +
  ggh4x::facet_nested(cols = vars(ext_lib_plate_nb), rows = vars(strain_group_qpcr, target), scales = "free", space = "free") +
  xlab("Well column") + ylab("Well row") 

Warning

From these visualizations of the raw data, something looks weird with the cq and copies_per_swab values for the first 3 plates of “VMRC group 1”.

Total number of copies per swab per sample type

g <- 
  qpcr |> 
  ggplot() +
  aes(x = target, y = copies_per_swab, col = qpcr_sample_type, fill = qpcr_sample_type) +
  geom_boxplot(alpha = 0.5) +
  theme(axis.text.x = element_text(angle = 45, hjust = 1)) +
  facet_grid(. ~ target_type, scales = "free", space = "free")

g 

g + scale_y_log10()

g <- 
  qpcr |> 
  ggplot() +
  aes(x = target, y = copies_per_swab, col = qpcr_sample_type, fill = qpcr_sample_type) +
  geom_boxplot(alpha = 0.5) +
  facet_grid(qpcr_sample_type ~ ., scales = "free") +
  guides(fill = "none", color = "none") +
  theme(
    axis.text.x = element_text(angle = 45, hjust = 1),
    strip.text.y = element_text(angle = 0)
  ) +
  facet_grid(. ~ target_type, scales = "free", space = "free")

g 

g + scale_y_log10()

g <- 
  qpcr |> 
  ggplot() +
  aes(x = ext_lib_plate_nb |> factor(), y = copies_per_swab, col = target, fill = target) +
  geom_boxplot(alpha = 0.5) +
  facet_grid(qpcr_sample_type ~ ., scales = "free") +
  xlab("Extraction plate number") +
  theme(
    strip.text.y = element_text(angle = 0),
    strip.text.x = element_text(angle = 90, hjust = 0)
  )

g 

g + scale_y_log10() 

Empty and water samples

From the plots above, it looks like the empty and water samples have non-zero values on a few LBP plates.

plot_qpcr_empties <- function(qpcr, color_by = "qpcr_sample_type") {
  qpcr |> 
    dplyr::filter(qpcr_sample_type %in% c("Empty", "Water")) |> 
    group_by(sample, pcr_plate_id, ext_lib_plate_nb, target) |>
    mutate(replicate_nb = row_number()) |> 
    ungroup() |> 
    ggplot() +
    aes(x = sample, y = copies_per_swab, label = replicate_nb, col = !!sym(color_by)) +
    geom_text(size = 3) +
    ggh4x::facet_nested(cols = vars(ext_lib_plate_nb), rows = vars(strain_group_qpcr, target), scales = "free_x", space = "free_x") +
    theme(
      axis.text.x = element_text(angle = 45, hjust = 1),
      strip.text.y = element_text(angle = 0)
    ) 
}

plot_qpcr_empties(qpcr, color_by = "qpcr_sample_type") 

plot_qpcr_empties(qpcr |> mutate(zero_copies = (copies_per_swab == 0)), color_by = "zero_copies") 

prob_target <-
  qpcr |> 
  select(target) |> 
  distinct() |> 
  mutate(prob_target = (target %in% c("C0022A1", "C0059E1", "C0175A1")))

Warning

We see the same issues as above for the first 3 plates of VMRC group 1.

Control samples

qpcr |> 
  dplyr::filter(qpcr_sample_type %in% c("Control sample")) |> 
  group_by(sample, pcr_plate_id, ext_lib_plate_nb, target) |>
  mutate(replicate_nb = row_number()) |> 
  ungroup() |> 
  mutate(
    control_type = control_type |> str_replace_na("? Unknown (not in MG manifest)"),
    vibr_sample_id = vibr_sample_id |> fct_reorder(control_type),
    plate_nb = str_c("ext. plate\n", ext_lib_plate_nb) |> fct_reorder(ext_lib_plate_nb),
    ) |> 
  ggplot() +
  aes(x = vibr_sample_id, y = copies_per_swab, label = replicate_nb, col = control_type) +
  geom_text(size = 3) +
  scale_y_log10() +
  scale_color_discrete("Control type") +
  ggh4x::facet_nested(cols = vars(plate_nb), rows = vars(strain_group_qpcr, target), scales = "free_x", space = "free_x") +
  theme(
    axis.text.x = element_text(angle = 45, hjust = 1),
    # strip.text.x = element_text(angle = 90),
    strip.text.y = element_text(angle = 0)
  ) 

These look consistent with expectations, except, again, for the first 3 plates of VMRC group 1.

Standards

qpcr |> 
  dplyr::filter(qpcr_sample_type %in% "Standard") |>
  ggplot() +
  aes(x = starting_quantity, y = cq, col = replicate_nb |> factor()) +
  geom_point(alpha = 0.5, size = 0.5) +
  ggh4x::facet_nested(cols = vars(ext_lib_plate_nb), rows = vars(strain_group_qpcr, target), scales = "free_x", space = "free_x") +
  scale_x_log10() +
  theme(
    strip.text.y = element_text(angle = 0),
    axis.text.x = element_text(angle = 45, hjust = 1)
  )

For the 16S, there seems to be “reverse saturation” (i.e., the cq is lower than expected with a linear relationship). Maybe the dilution was not that reliable at such low concentrations.

Target C0022A1

Target C0059E1

Target C0175A1

Target FF00018

Target FF00051

Target UC101

Target 185329

Target C0028A1

Target C0112A1

Target 122010

Target C0006A1

Target FF00004

Target FF00064

Target FF00072

Target UC119

Target 16S

qpcr |> 
  dplyr::filter(qpcr_sample_type %in% "Standard") |>
  mutate(std_nb = content |> str_remove("Std-") |> parse_number()) |> 
  ggplot() +
  aes(x = starting_quantity, y = cq, col = dilution_factor |> factor()) +
  geom_path(aes(group = interaction(ext_lib_plate_nb, replicate_nb)), alpha = 0.2) +
  geom_text(alpha = 0.2, aes(label = std_nb)) +
  ggh4x::facet_nested_wrap(vars(strain_group_qpcr, target), ncol = 9) +
  scale_x_log10() +
  scale_color_manual(
    name = "Dilution factor",
    breaks = c(5,10,20,30),
    values = c("black", "deeppink3", "deeppink1", "pink")
  ) +
  theme(
    axis.text.x = element_text(angle = 45, hjust = 1)
  )

It looks like standard 4 was not prepared or placed properly for VMRC group 4.

qpcr |> 
  dplyr::filter(qpcr_sample_type %in% "Standard") |>
  mutate(std_nb = content |> str_remove("Std-") |> parse_number()) |> 
  ggplot() +
  aes(x = starting_quantity, y = cq, col = ext_lib_plate_nb |> factor()) +
  geom_path(aes(group = interaction(ext_lib_plate_nb, replicate_nb)), alpha = 0.2) +
  geom_text(alpha = 0.2, aes(label = std_nb)) +
  ggh4x::facet_nested_wrap(vars(strain_group_qpcr, target), ncol = 9) +
  scale_x_log10() +
  theme(
    axis.text.x = element_text(angle = 45, hjust = 1)
  )

This shows again the issues with the first 3 plates of VMRC group 1.

Clinical samples

qpcr |> 
  dplyr::filter(qpcr_sample_type %in% c("VIBRANT clinical sample")) |> 
  group_by(vibr_sample_id, sample, pcr_plate_id, ext_lib_plate_nb, target) |>
  mutate(
    replicate_nb = row_number(),
    median_cps = median(copies_per_swab, na.rm = TRUE),
    mean_cps = mean(copies_per_swab, na.rm = TRUE)
    ) |> 
  ungroup() |> 
  mutate(vibr_sample_id = vibr_sample_id |> fct_reorder(mean_cps)) |>
  ggplot() +
  aes(x = vibr_sample_id, y = copies_per_swab, label = replicate_nb, col = replicate_nb |> factor()) +
  geom_line(aes(group = vibr_sample_id), col = "black", linewidth = 0.1) +
  geom_text(size = 3) +
  scale_y_log10() +
  scale_color_discrete("replicate") +
  scale_x_discrete("Samples", breaks = NULL) +
  ggh4x::facet_nested(cols = vars(ext_lib_plate_nb), rows = vars(strain_group_qpcr, target), scales = "free_x", space = "free_x") +
  theme(
    axis.text.x = element_text(angle = 45, hjust = 1),
    strip.text.y = element_text(angle = 0)
  )

We notice again the same issues with the VRMC group 1 plate 1-3.

Warning

We could simply discard these values (exclude these targets x plates) or try to identify a new threshold to differentiate between background noise and actual signal.

Replicates

qpcr |> 
  dplyr::filter(qpcr_sample_type %in% c("VIBRANT clinical sample")) |> 
  group_by(qpcr_sample_id, vibr_sample_id, target) |>
  mutate(
    n_replicates = n(),
    replicate_nb = row_number(),
    median_cps = median(copies_per_swab, na.rm = TRUE),
    mean_cps = mean(copies_per_swab, na.rm = TRUE),
    min_ext_lib_plate_nb = min(ext_lib_plate_nb, na.rm = TRUE),
    ) |> 
  ungroup() |> 
  dplyr::filter(n_replicates > 3) |>
  mutate(vibr_sample_id = vibr_sample_id |> fct_reorder(mean_cps)) |>
  ggplot() +
  aes(x = ext_lib_plate_nb |> factor(), y = copies_per_swab, label = replicate_nb, col = (ext_lib_plate_nb == 12)) +
  ggh4x::facet_nested(cols = vars(min_ext_lib_plate_nb), rows = vars(strain_group_qpcr, target), scales = "free_x", space = "free_x") +
  geom_text(size = 3) +
  scale_y_log10() +
  scale_color_discrete("replicate") +
  scale_x_discrete("Extraction plate") +
  theme(
    axis.text.x = element_text(angle = 90, hjust = 1),
    strip.text.y = element_text(angle = 0),
  )

Agreement between pairs of triplicates is good except for the samples that initially were on the first 3 plates for VMRC group 1.

New detection thresholds ?

The idea is to use the negative controls cq values to define plate- and target-specific “detection” thresholds, then use these thresholds to set to 0 the copies_per_swab values for which the cq values are above the threshold.

The figures below show the cq values for the VMRC group 1 and 2.

qpcr |>
  dplyr::filter(strain_group_qpcr %in% c("VMRC_1", "VMRC_2")) |>
  ggplot() +
  aes(
    x = well_row |> factor(), 
    y = well_col |> fct_rev(), 
    fill = cq
    ) +
  geom_tile() +
  ggh4x::facet_nested(cols = vars(ext_lib_plate_nb), rows = vars(strain_group_qpcr, target), scales = "free", space = "free") +
  xlab("Well column") + ylab("Well row") 

We impute the missing cq values to 45 (well above the maximum observed value, and what I assume was the total number of cycles).

qpcr <- qpcr |> mutate(cq_imp = cq |> replace_na(45)) 

qpcr |>
  dplyr::filter(strain_group_qpcr %in% c("VMRC_1", "VMRC_2")) |>
  ggplot() +
  aes(
    x = well_row |> factor(), 
    y = well_col |> fct_rev(), 
    fill = cq_imp
    ) +
  geom_tile() +
  ggh4x::facet_nested(cols = vars(ext_lib_plate_nb), rows = vars(strain_group_qpcr, target), scales = "free", space = "free") +
  xlab("Well column") + ylab("Well row") 

Comparing the cq values per sample type

qpcr <- 
  qpcr |>
  mutate(
    simplified_type = 
      case_when(
        str_detect(control_type, "Mock") ~ "Positive control",
        (qpcr_sample_type == "Control sample") & is.na(sample_type) ~ "Unknown control",
        qpcr_sample_type %in% c("Control sample", "Empty", "Water") ~ "Negative control",
        TRUE ~ qpcr_sample_type
      ) |> 
      fct_infreq(),
    qpcr_sample_type = qpcr_sample_type |> fct_infreq()
    ) 

# qpcr |> dplyr::count(simplified_type, sample_type, control_type, qpcr_sample_type)

We define the thresholds as the average over the two smallest cq value for the “empties” (i.e., empty and water samples, as well as the negative controls).

thresholds <- 
  qpcr |> 
  dplyr::filter(target != "16S") |> 
  group_by(target, strain_group_qpcr, ext_lib_plate_nb) |> 
  summarize(
    min_cq_empties = min(cq_imp[simplified_type == "Negative control"], na.rm = TRUE),
    robust_min_cq_empties = cq_imp[simplified_type == "Negative control"] |> sort() |> extract(1:2) |> mean(),
    max_cq_std = max(cq_imp[simplified_type == "Standard"], na.rm = TRUE),
    .groups = "drop"
  ) |> 
  mutate(
    threshold_cq = robust_min_cq_empties
  )

qpcr |> 
  dplyr::filter(
    strain_group_qpcr %in% c("VMRC_1", "VMRC_2"), 
    ext_lib_plate_nb <= 6,
    simplified_type %in% c("VIBRANT clinical sample", "Standard", "Negative control") 
    ) |> 
  arrange(simplified_type) |> 
  ggplot() +
  aes(x = simplified_type |> str_wrap(width = 15), y = cq_imp, col = qpcr_sample_type) + #  
  geom_violin(col = "gray") +
  geom_jitter(height = 0, width = 0.25, alpha = 0.5, size = 0.75) +
  geom_hline(
    data = thresholds |> dplyr::filter(strain_group_qpcr %in% c("VMRC_1", "VMRC_2"), ext_lib_plate_nb <= 6), 
    aes(yintercept = threshold_cq), col = "steelblue1"
    ) +
  facet_grid(target ~ ext_lib_plate_nb) +
  scale_color_manual(values = c("steelblue1", "black", "dodgerblue3", "red","pink") |> rev()) +
  theme(
    axis.text.x = element_text(angle = 90, hjust = 1, vjust = 0.5)
  ) +
  xlab("Sample type") 

qpcr <- 
  qpcr |> 
  select(-any_of(c("min_cq_empties","robust_min_cq_empties", "max_cq_std", "threshold_cq"))) |> 
  dplyr::left_join(thresholds, by = join_by(ext_lib_plate_nb, strain_group_qpcr, target)) 

For all plates and targets

qpcr |>
  dplyr::filter(qpcr_sample_type == "VIBRANT clinical sample") |> 
  ggplot() +
  aes(x = cq_imp, y = target) +
  geom_violin(fill = "gray90", col = "transparent") +
  geom_jitter(width = 0, size = 0.1, col = "pink") +
  geom_vline(aes(xintercept = min_cq_empties), col = "dodgerblue") +
  geom_vline(aes(xintercept = robust_min_cq_empties), col = "green3") +
  geom_vline(aes(xintercept = max_cq_std), col = "red") +
  ggh4x::facet_nested(cols = vars(ext_lib_plate_nb), rows = vars(strain_group_qpcr, target), scales = "free_y", space = "free_y") +
  theme(strip.text.y = element_text(angle = 0)) +
  labs(
    x = "cq value (imputed to 45 when not avalaible)",
    caption = 
      str_c(
        "Pink dots are the cq values for the VIBRANT clinical samples\n",
        "The blue lines are the minimum cq value for the negative controls\n",
        "The green lines are the average of the two smallest cq values for the negative controls (= the detection threshold)\n",
        "The red lines are the maximum cq value for the standards"
      )
  )

Adjusted copies per swabs

We create a new column copies_per_swab_adj that is set to 0 if the cq_imp is above the threshold.

qpcr <- 
  qpcr |> 
  mutate(
    copies_per_swab_adj = 
      case_when(
        (target != "16S") & (cq_imp >= threshold_cq) ~ 0,
        TRUE ~ copies_per_swab
      )
  )

qpcr |> 
  dplyr::filter(qpcr_sample_type %in% c("VIBRANT clinical sample")) |> 
  group_by(qpcr_sample_id, sample, pcr_plate_id, ext_lib_plate_nb, target) |>
  mutate(
    replicate_nb = row_number(),
    median_cps = median(copies_per_swab_adj, na.rm = TRUE),
    mean_cps = mean(copies_per_swab_adj, na.rm = TRUE)
    ) |> 
  ungroup() |> 
  mutate(qpcr_sample_id = qpcr_sample_id |> fct_reorder(mean_cps)) |>
  ggplot() +
  aes(x = qpcr_sample_id, y = copies_per_swab_adj, label = replicate_nb, col = replicate_nb |> factor()) +
  geom_line(aes(group = qpcr_sample_id), col = "black", linewidth = 0.1) +
  geom_text(size = 3) +
  scale_y_log10() +
  scale_color_discrete("replicate") +
  scale_x_discrete("Samples", breaks = NULL) +
  ggh4x::facet_nested(cols = vars(ext_lib_plate_nb), rows = vars(strain_group_qpcr, target), scales = "free_x", space = "free_x") +
  theme(
    axis.text.x = element_text(angle = 45, hjust = 1),
    strip.text.y = element_text(angle = 0)
  )

This does not fully solve the problem, but I hope that at the “aggregation” step, taking the median value would remove a lot of false positive.

If not, a more stringent threshold could potentially be used, or these samples would have to be excluded from the analysis.

Aggregation across replicates

qpcr <- 
  qpcr |> 
  group_by(qpcr_sample_id, target) |> 
  mutate(
    copies_per_swab_raw_median = median(copies_per_swab, na.rm = TRUE),
    copies_per_swab_adj_median = median(copies_per_swab_adj, na.rm = TRUE),
    copies_per_swab_adj_range = max(copies_per_swab_adj, na.rm = TRUE) - min(copies_per_swab_adj, na.rm = TRUE),
  ) |> 
  ungroup()

qpcr |> 
  dplyr::filter(qpcr_sample_type %in% c("VIBRANT clinical sample")) |>
  group_by(qpcr_sample_id) |> mutate(n_target_detected = sum(copies_per_swab_raw_median > 0)) |> ungroup() |>
  mutate(qpcr_sample_id = qpcr_sample_id |> fct_reorder(n_target_detected)) |>
  ggplot() +
  aes(x = qpcr_sample_id, y = target, fill = copies_per_swab_raw_median |> log10()) +
  geom_tile() +
  facet_grid(strain_group_qpcr + LBP  ~ ext_lib_plate_nb, scales = "free", space = "free") +
  scale_fill_gradient(low = "dodgerblue1", high = "black", na.value = "white") +
  theme(
    axis.text.x = element_blank(),
    legend.position = "bottom"
  ) +
  ggtitle("Median of the original `copies_per_swabs`")

qpcr |> 
  dplyr::filter(qpcr_sample_type %in% c("VIBRANT clinical sample")) |>
  group_by(qpcr_sample_id) |> mutate(n_target_detected = sum(copies_per_swab_adj_median > 0)) |> ungroup() |>
  mutate(qpcr_sample_id = qpcr_sample_id |> fct_reorder(n_target_detected)) |>
  ggplot() +
  aes(x = qpcr_sample_id, y = target, fill = copies_per_swab_adj_median |> log10()) +
  geom_tile() +
  facet_grid(strain_group_qpcr + LBP  ~ ext_lib_plate_nb, scales = "free", space = "free") +
  scale_fill_gradient(low = "dodgerblue1", high = "black", na.value = "white") +
  theme(
    axis.text.x = element_blank(),
    legend.position = "bottom"
  ) +
  ggtitle("Median of the adjusted `copies_per_swabs`")

The threshold correction likely helped in reducing the number of false positive.

Longitudinal profiles

When looking at the longitudinal patterns for randomized participants, we do not notice an over-representation of a specific plate or group in the “isolated” positives:

qpcr |> 
  dplyr::filter(
    sample_type %in% c("Clinical sample"), 
    # sample_category %in% c("Expected sample"),
    mg_visit_code %in% c(seq(1000, 1900, by = 100), 2120)
  ) |>
  group_by(mg_pid) |> mutate(n_target_detected = sum(copies_per_swab_adj_median[!is.na(LBP)] > 0)) |> ungroup() |>
  mutate(mg_pid = mg_pid |> fct_reorder(n_target_detected)) |>
  ggplot() +
  aes(
    x = mg_pid, y = mg_visit_code, 
    fill = ext_lib_plate_nb |> factor(), 
    alpha = copies_per_swab_adj_median |> log10()
  ) +
  geom_tile() +
  ggh4x::facet_nested(rows = vars(strain_group_qpcr, target), scales = "free", space = "free") +
  scale_fill_discrete("Extraction plate number") +
  scale_alpha_continuous(limits = c(0, 10)) +
  theme(
    axis.text.x = element_text(angle = 90, hjust = 1, vjust = 0.5),
    strip.text.y = element_text(angle = 0),
    legend.position = "bottom"
  )

We should thus expect some false positive in the LBP qPCR data :)

qpcr |> 
  dplyr::filter(
    target != "16S",
    sample_type %in% c("Clinical sample"), 
    mg_visit_code %in% c(0, seq(1000, 1900, by = 100), 2120)
  ) |>
  # group_by(mg_pid) |> mutate(n_target_detected = sum(copies_per_swab_adj_median[!is.na(LBP)] > 0)) |> ungroup() |>
  # mutate(mg_pid = mg_pid |> fct_reorder(n_target_detected)) |>
  mutate(mg_pid = mg_pid |> factor()) |>
  mutate(target = target |> fct_reorder(as.numeric(factor(LBP)) + as.numeric(factor(strain_origin))/10)) |> 
  ggplot() +
  aes(
    x = target, y = mg_pid |> fct_rev(), 
    fill = LBP,
    alpha = copies_per_swab_adj_median |> log10()
  ) +
  geom_tile() +
  facet_grid(. ~ mg_visit_code) +
  theme(
    axis.text.x = element_text(angle = 90, hjust = 1, vjust = 0.5),
    # strip.text.y = element_text(angle = 0),
    legend.position = "bottom"
  )

There are clear patterns in the data, hinting at the participants’ arms :) However, this also suggests that there might be quite a lot of false positive and a low specificity for the qPCR probes.

qpcr |> 
  dplyr::filter(
    sample_type %in% c("Clinical sample"), 
    # sample_category %in% c("Expected sample"),
    mg_visit_code %in% c(seq(1000, 1900, by = 100), 2120),
    target == "16S"
    ) |>  
  arrange(mg_pid, mg_visit_code) |> 
  select(mg_pid, mg_visit_code, copies_per_swab_adj_median) |>
  distinct() |> 
  ggplot() +
  aes(x = mg_visit_code, y = copies_per_swab_adj_median, col = mg_pid) +
  geom_boxplot(alpha = 0.2, fill = "dodgerblue1", color = "dodgerblue1") +
  geom_line(aes(group = mg_pid), alpha = 0.2) +
  # geom_point(size = 0.5) +
  ggbeeswarm::geom_quasirandom(width = 0.2) +
  scale_y_log10() +
  guides(col = "none") +
  ggtitle("16S copies per swab") 

Specificity and sensitivity estimation from Mock positive controls and from screening visits samples

In this section, we use the biological controls (Mock samples) to estimate the sensitivity (true positive rate) of the qPCR assays and the screening visit samples to estimate the specificity (false positive rate).

Our true positive samples are the Mock samples since they contain, among other strains, the 15 LBP strains.

The relative abundance of each LBP strains in Mock 1 samples should be around 1/100, while in Mock 2 samples, it should be around 1/24.

Unfortunately, we do not have a good biological negative sample (e.g., a pool of non-LBP strains), so we use the screening visit samples as our “true negative samples”, acknowledging that some of these samples might contain native L. crispatus that may be very closely related to one of the LBP strains.

tmp <- 
  qpcr |> 
  dplyr::filter(
    (sample_type == "Positive control") | ((mg_visit_code <= 1000) & (sample_type == "Clinical sample"))
  ) |> 
  select(
    uid, mg_visit_code, sample_type, control_type, ext_lib_plate_nb, target, strain_group_qpcr, LBP, strain_origin, copies_per_swab_adj_median
  ) |> 
  arrange(uid) |> 
  distinct() |> 
  mutate(
    truth = 
      case_when(
        str_detect(control_type, "Mock") ~ "P",
        (mg_visit_code <= 1000) ~ "N",
        TRUE ~ NA_character_
      ),
    predicted = 
      case_when(
        copies_per_swab_adj_median > 0 ~ "PP",
        TRUE ~ "PN"
      )
  )

tmp |> 
  ggplot() +
  aes(
    x = sample_type, y = copies_per_swab_adj_median |> asinh(), 
    col = str_c(sample_type, " ",control_type |> as.character(), " (", truth, ")")
    ) +
  ggh4x::facet_nested(cols = vars(strain_group_qpcr, target)) +
  scale_color_discrete("Control type") + xlab("") +
  ggbeeswarm::geom_quasirandom(size = 0.7, alpha = 0.8) +
  theme(
    #strip.text.x = element_text(angle = 90),
    axis.text.x = element_blank()
  )

tmp <- 
  tmp |> 
  dplyr::filter(target != "16S") |> 
  dplyr::left_join(
    tmp |> 
      dplyr::filter(target == "16S") |> 
      select(uid, copies_per_swab_adj_median) |> 
      dplyr::rename(copies_per_swab_adj_median_16S = copies_per_swab_adj_median),
    by = join_by(uid)
  )

tmp <- 
  tmp |> 
  mutate(
    exp_rel_ab = 
      case_when(
        (control_type == "Mock 1") ~ 1 / 100,
        (control_type == "Mock 2") ~ 1 / 24,
        TRUE ~ 0
      ),
    obs_rel_ab = copies_per_swab_adj_median / copies_per_swab_adj_median_16S
  )

g <- 
  tmp |> 
  ggplot() +
  aes(
    x = str_c(LBP, strain_origin, target, sep = " - "), y = obs_rel_ab, 
    col = str_c(sample_type, " ", control_type, " (", truth, ")")
  ) +
  facet_grid(.  ~ sample_type + control_type) + # , scales = "free"
  geom_hline(aes(yintercept = exp_rel_ab), col = "gray50") +
  geom_boxplot(alpha = 0, outlier.shape = NA) +
  geom_point(alpha = 0.3) +
  scale_color_discrete("Control type") + 
  ylab("Observed relative abundance") + 
  xlab("LBP strain") + 
  labs(
    caption = "The horizontal lines are the expected relative abundance of each LBP strain in the control samples."
  ) +
  theme(
    axis.text.x = element_text(angle = 90, vjust = 0.5, hjust = 1)
  )

g

g + ylim(c(0, 0.25)) 

g + ylim(c(0, 0.05)) 

tmp |> 
  group_by(target, strain_group_qpcr, LBP, strain_origin) |>
  summarize(
    `n True Negatives` = sum(truth == "N" & predicted == "PN"),
    `n Negatives` = sum(truth == "N"),
    specificity = `n True Negatives` / `n Negatives`,
    `n True Positives` = sum(truth == "P" & predicted == "PP"),
    `n Positives` = sum(truth == "P"),
    sensitivity = sum(truth == "P" & predicted == "PP") / sum(truth == "P"),
    .groups = "drop"
  ) |> 
  arrange(strain_group_qpcr, target) |> 
  gt(caption = "Specificity and sensitivity of the qPCR assays for the LBP targets") |>
  cols_label(
    specificity ~ "Specificity (%) [likely underestimated]",
    sensitivity ~ "Sensitivity (%)",
    strain_origin ~ "Strain origin",
    strain_group_qpcr ~ "qPCR group",
    target ~ "Target (LBP strain)"
  ) |>
  fmt_number(
    columns = c(specificity, sensitivity),
    decimals = 2,
    scale_by = 100
  ) 

Specificity and sensitivity of the qPCR assays for the LBP targets

Target (LBP strain)	qPCR group	LBP	Strain origin	n True Negatives	n Negatives	Specificity (%) [likely underestimated]	n True Positives	n Positives	Sensitivity (%)
C0022A1	VMRC_1	LC-106 & LC-115	US	290	296	97.97	21	22	95.45
C0059E1	VMRC_1	LC-106 & LC-115	US	274	296	92.57	21	22	95.45
C0175A1	VMRC_1	LC-106 & LC-115	US	281	296	94.93	21	22	95.45
FF00018	VMRC_2	LC-106 & LC-115	SA	286	296	96.62	21	22	95.45
FF00051	VMRC_2	LC-106 & LC-115	SA	278	296	93.92	21	22	95.45
UC101	VMRC_2	LC-106 & LC-115	SA	284	296	95.95	21	22	95.45
185329	VMRC_3	LC-115	SA	296	296	100.00	15	22	68.18
C0028A1	VMRC_3	LC-115	US	294	296	99.32	13	22	59.09
C0112A1	VMRC_3	LC-115	US	294	296	99.32	21	22	95.45
122010	VMRC_4	LC-115	SA	284	296	95.95	21	22	95.45
C0006A1	VMRC_4	LC-115	US	291	296	98.31	21	22	95.45
FF00004	VMRC_4	LC-115	SA	293	296	98.99	21	22	95.45
FF00064	VMRC_5	LC-115	SA	291	296	98.31	21	22	95.45
FF00072	VMRC_5	LC-115	SA	295	296	99.66	21	22	95.45
UC119	VMRC_5	LC-115	SA	296	296	100.00	21	22	95.45

tmp |> 
  dplyr::filter(sample_type == "Clinical sample") |> 
  mutate(
    site = case_when(
      str_detect(uid, "06810") ~ "MGH",
      str_detect(uid, "06820") ~ "CAP",
      TRUE ~ "Other"
    )
  ) |> 
  ggplot() +
  aes(
    x = site, y = copies_per_swab_adj_median |> asinh(), 
    col = site
    ) +
  ggh4x::facet_nested(cols = vars(strain_group_qpcr, target)) +
  scale_color_manual("Study site", values = site_colors) + 
  ggbeeswarm::geom_quasirandom(size = 0.7, alpha = 0.8) +
  theme(
    #strip.text.x = element_text(angle = 90),
    axis.text.x = element_text(angle = 90, hjust = 1, vjust = 0.5)
  )

It looks like there might be a mild correlation between strain origin and study site for screening visit samples.

Creating SummarizedExperiment objects

We create two Summarized Experiment objects:

• one with all values as provided in the original file, where each “sample” is a plate well and each feature is a target (LBP taxa)

• one with summarized values (aggregated across replicates) where each “sample” is a VIBRANT sample ID (including “biological” control samples) and each feature is a target (LBP taxa)

Raw SummarizedExperiment object

We create a SummarizedExperiment object with the following assays:

• dilution_factor
• cq
• starting_quantity
• cq_mean
• quant_adjusted
• copies_per_swab_raw (current copies_per_swab column)
• copies_per_swab (current copies_per_swab_adj column)

The “samples” are defined by the qpcr_uid column (one per well and plate) and the “features” are defined by the target column (i.e., the LBP taxa).

The colData contains the following columns:

• qpcr_uid (the qPCR specific unique identifier)
• uid (the VIBRANT cross-assay unique sample identifier)
• qpcr_sample_id
• vibr_sample_id
• qpcr_sample_type
• sample_type
• control_type
• ext_lib_plate_nb
• ext_lib_plate_id

The rowData contains the following columns:

• fluor
• strain_group_qpcr
• pcr_plate_id (the concatenation of pcr_plate_id for each ext_lib_plate_nb)
• taxon_label
• LBP
• strain_id
• strain_origin
• biose_id

qpcr |> dplyr::count(qpcr_uid) |> dplyr::count(n)

qpcr |> dplyr::count(qpcr_uid) |> nrow()


qpcr |> dplyr::count(qpcr_uid) |> dplyr::filter(n != 16) |> View()

qpcr |> 
  select(
    qpcr_uid, 
    uid, mg_pid, mg_visit_code,
    qpcr_sample_type,  sample_type, control_type, 
    qpcr_sample_id, vibr_sample_id, replicate_nb, 
    ext_lib_plate_nb, ext_lib_plate_id
  ) |> 
  distinct() |> 
  group_by(qpcr_uid) |> mutate(n = n()) |> ungroup() |> 
  dplyr::filter(n > 1) |> 
  arrange(qpcr_uid)

qpcr |> 
  dplyr::filter(qpcr_sample_id == "1588") |> 
  select(qpcr_sample_id, qpcr_uid, pcr_plate_id, well_row, well_col, ext_lib_plate_nb, ext_lib_plate_id, target) |> 
  arrange(target, pcr_plate_id,  qpcr_uid, well_row, well_col) |> 
  View()

make_raw_qpcr_SE <- function(qpcr){
  
  qpcr <- 
    qpcr |> 
    arrange(ext_lib_plate_nb, well_col, well_row) |>
    mutate(qpcr_uid = qpcr_uid |> fct_inorder()) |> 
    arrange(strain_group_qpcr, fluor) |> 
    mutate(target = target |> fct_inorder()) |> 
    arrange(qpcr_uid, target)
    
  # ASSAYS
  
  dilution_assay <- 
    qpcr |>
    select(qpcr_uid, target, dilution_factor) |> 
    arrange() |> 
    pivot_wider(names_from = qpcr_uid, values_from = dilution_factor) |> 
    as.data.frame() |> 
    column_to_rownames("target") 
  
  cq_assay <- 
    qpcr |>
    select(qpcr_uid, target, cq) |> 
    arrange() |> 
    pivot_wider(names_from = qpcr_uid, values_from = cq) |> 
    as.data.frame() |> 
    column_to_rownames("target") 
  
  starting_quantity_assay <- 
    qpcr |>
    select(qpcr_uid, target, starting_quantity) |> 
    arrange() |> 
    pivot_wider(names_from = qpcr_uid, values_from = starting_quantity) |> 
    as.data.frame() |> 
    column_to_rownames("target")
  
   cq_mean_assay <- 
    qpcr |>
    select(qpcr_uid, target, cq_mean) |> 
    arrange() |> 
    pivot_wider(names_from = qpcr_uid, values_from = cq_mean) |> 
    as.data.frame() |> 
    column_to_rownames("target")
   
   quant_adjusted_assay <- 
     qpcr |>
     select(qpcr_uid, target, quant_adjusted) |> 
     arrange() |> 
     pivot_wider(names_from = qpcr_uid, values_from = quant_adjusted) |> 
     as.data.frame() |> 
     column_to_rownames("target")
   
   copies_per_swab_raw_assay <- 
     qpcr |>
     select(qpcr_uid, target, copies_per_swab) |> 
     arrange() |> 
     pivot_wider(names_from = qpcr_uid, values_from = copies_per_swab) |> 
     as.data.frame() |> 
     column_to_rownames("target")
   
  copies_per_swab_assay <- 
    qpcr |>
    select(qpcr_uid, target, copies_per_swab_adj) |> 
    arrange() |> 
    pivot_wider(names_from = qpcr_uid, values_from = copies_per_swab_adj) |> 
    as.data.frame() |> 
    column_to_rownames("target")
  
    # COLDATA
   coldata <- 
     qpcr |> 
     select(
       qpcr_uid, 
       uid, mg_pid, mg_visit_code,
       qpcr_sample_type,  sample_type, control_type, 
       qpcr_sample_id, vibr_sample_id, replicate_nb, 
       ext_lib_plate_nb #, ext_lib_plate_id
       ) |> 
     distinct() |> 
     arrange(qpcr_uid) |> 
     as.data.frame() |> 
     mutate(rownames = qpcr_uid) |> 
     column_to_rownames("rownames") 
  

    # ROWDATA
   rowdata <-
     qpcr |> 
     select(
       target, fluor, strain_group_qpcr, 
       pcr_plate_id, ext_lib_plate_nb, 
       taxon_label, LBP, strain_id, strain_origin, biose_id
       ) |> 
     distinct() |> 
     group_by(
       target, fluor, strain_group_qpcr, 
       taxon_label, LBP, strain_id, strain_origin, biose_id
       ) |> 
     arrange(target, ext_lib_plate_nb) |> 
     mutate(
       pcr_plate_id = str_c(pcr_plate_id, " (extr. plate ", ext_lib_plate_nb |> str_pad(width = 2, pad = "0"), ")", sep = ""),
     ) |> 
     summarize(
       pcr_plate_ids = pcr_plate_id |> unique() |> str_c(collapse = ", "),
       .groups = "drop"
     ) |> 
     ungroup() |> 
     as.data.frame() |> 
     mutate(rownames = target) |> 
     column_to_rownames("rownames")

  
   
   SE <- SummarizedExperiment(
     assays = list(
       dilution = dilution_assay |> as.matrix(),
       cq = cq_assay |> as.matrix(),
       cq_mean = cq_mean_assay |> as.matrix(),
       starting_quantity = starting_quantity_assay |> as.matrix(),
       quant_adjusted = quant_adjusted_assay |> as.matrix(),
       copies_per_swab_raw = copies_per_swab_raw_assay |>  as.matrix(),
       copies_per_swab = copies_per_swab_assay |> as.matrix()
     ),
     colData = coldata,
     rowData = rowdata,
     metadata = list(
       description = "Raw qPCR data from the VIBRANT study",
       date = today(),
       assay_and_coldata_dictionary = dictionary
     )
   )
   
   SE
  
}

SE_qPCR_raw <- make_raw_qpcr_SE(qpcr)
SE_qPCR_raw

# A SummarizedExperiment-tibble abstraction: 63,312 × 29
# Features=16 | Samples=3957 | Assays=dilution, cq, cq_mean, starting_quantity,
#   quant_adjusted, copies_per_swab_raw, copies_per_swab
   .feature .sample      dilution    cq cq_mean starting_quantity quant_adjusted
   <chr>    <chr>           <dbl> <dbl>   <dbl>             <dbl>          <dbl>
 1 C0175A1  std_02_plat…       10  18.5    18.5           1000000       10000000
 2 C0022A1  std_02_plat…       10  19.3    19.3           1000000       10000000
 3 C0059E1  std_02_plat…       10  18.8    18.8           1000000       10000000
 4 UC101    std_02_plat…       10  17.3    17.2           1000000       10000000
 5 FF00018  std_02_plat…       10  18.2    18.1           1000000       10000000
 6 FF00051  std_02_plat…       10  18.1    18.1           1000000       10000000
 7 185329   std_02_plat…       30  17.1    17.1           1000000       30000000
 8 C0028A1  std_02_plat…       30  17.2    17.2           1000000       30000000
 9 C0112A1  std_02_plat…       30  17.8    17.8           1000000       30000000
10 FF00004  std_02_plat…       10  18.0    18.1           1000000       10000000
# ℹ 310 more rows
# ℹ 22 more variables: copies_per_swab_raw <dbl>, copies_per_swab <dbl>,
#   qpcr_uid <fct>, uid <chr>, mg_pid <chr>, mg_visit_code <chr>,
#   qpcr_sample_type <fct>, sample_type <chr>, control_type <chr>,
#   qpcr_sample_id <chr>, vibr_sample_id <chr>, replicate_nb <int>,
#   ext_lib_plate_nb <dbl>, target <fct>, fluor <chr>, strain_group_qpcr <chr>,
#   taxon_label <chr>, LBP <fct>, strain_id <fct>, strain_origin <fct>, …

Aggregated SummarizedExperiment object

We create a SummarizedExperiment object with summarized values (aggregated across replicates) where each “sample” is a VIBRANT sample ID (including control samples) and each feature is a target (LBP taxa), with the following assays

• dilution
• copies_per_swab_med (the median copies_per_swab across replicates)
• copies_per_swab_mean (the mean copies_per_swab across replicates)
• copies_per_swab_cv (the coefficient of variation of copies_per_swab across replicates)

The colData contains the same columns as above (except for those that are replicate-specific)

The rowData contains the same columns as above,

agg_qpcr_SE <- function(SE_qPCR_raw){
  
  filtered_qpcr <- 
    SE_qPCR_raw |> 
    dplyr::filter(
      !(qpcr_sample_type %in% c("Standard", "Water", "Empty")),
      !is.na(uid) 
      ) 
    
  summarized_qpcr <- 
    filtered_qpcr |>
    as_tibble() |> 
    group_by(uid, target) |> 
    summarize(
      n_non_na = sum(!is.na(copies_per_swab)),
      dilution = mean(dilution, na.rm = TRUE),
      copies_per_swab_med = median(copies_per_swab, na.rm = TRUE),
      copies_per_swab_mean = mean(copies_per_swab, na.rm = TRUE),
      copies_per_swab_sd = sd(copies_per_swab, na.rm = TRUE),
      copies_per_swab_range = max(copies_per_swab, na.rm = TRUE) - min(copies_per_swab, na.rm = TRUE),
      .groups = "drop"
    ) |> 
    mutate(
      copies_per_swab_cv = ifelse(copies_per_swab_sd == 0, 0, copies_per_swab_sd / copies_per_swab_mean)
    ) 

  # COLDATA
  coldata <- 
    filtered_qpcr |> 
    colData() |> 
    as.data.frame() |> 
    as_tibble() |> 
    select(-qpcr_uid, -replicate_nb, -starts_with("ext_lib")) |> 
    distinct() |>  
    arrange(uid)
  
  coldata <-  
    coldata |> 
    dplyr::left_join(
      filtered_qpcr |> 
        colData() |> 
        as.data.frame() |> 
        as_tibble() |> 
        select(uid, starts_with("ext_lib")) |> 
        group_by(uid) |> 
        summarize(
          ext_lib_plate_nb = str_c(ext_lib_plate_nb |> unique() |> sort(), collapse = ", "),
          # ext_lib_plate_id = str_c(ext_lib_plate_id |> str_replace_na("unknown") |> unique() |> sort(), collapse = ", "),
          .groups = "drop"
        ),
      by = join_by(uid)
    )
  
   coldata <-  
    coldata |> 
    as.data.frame() |> 
    mutate(rownames = uid) |> 
    column_to_rownames("rownames")
  
  # ROWDATA
   rowdata <-
     filtered_qpcr |> 
     rowData() |> 
     as.data.frame()
    
   SE <- SummarizedExperiment(
     assays = list(
       n_non_na = 
         summarized_qpcr |> 
         pivot_wider(id_cols = target, names_from = uid, values_from = n_non_na) |> 
         as.data.frame() |> 
         column_to_rownames("target") |> 
         as.matrix(),
        dilution = 
         summarized_qpcr |> 
         pivot_wider(id_cols = target, names_from = uid, values_from = dilution) |> 
         as.data.frame() |> 
         column_to_rownames("target") |> 
         as.matrix(),
       copies_per_swab_med = 
         summarized_qpcr |> 
         pivot_wider(id_cols = target, names_from = uid, values_from = copies_per_swab_med) |> 
         as.data.frame() |> 
         column_to_rownames("target") |> 
         as.matrix(),
       copies_per_swab_mean = 
         summarized_qpcr |> 
         pivot_wider(id_cols = target, names_from = uid, values_from = copies_per_swab_mean) |> 
         as.data.frame() |> 
         column_to_rownames("target") |> 
         as.matrix(),
       copies_per_swab_cv = 
         summarized_qpcr |> 
         pivot_wider(id_cols = target, names_from = uid, values_from = copies_per_swab_cv) |> 
         as.data.frame() |> 
         column_to_rownames("target") |> 
         as.matrix()
     ),
     colData = coldata,
     rowData = rowdata,
     metadata = list(
       description = "Aggregated qPCR data from the VIBRANT study",
       date = today(),
       assay_and_coldata_dictionary = SE_qPCR_raw@metadata$assay_and_coldata_dictionary
     )
   )
   
   SE
     
}

SE_qPCR_agg <- agg_qpcr_SE(SE_qPCR_raw = SE_qPCR_raw)
SE_qPCR_agg

# A SummarizedExperiment-tibble abstraction: 16,464 × 25
# Features=16 | Samples=1029 | Assays=n_non_na, dilution, copies_per_swab_med,
#   copies_per_swab_mean, copies_per_swab_cv
   .feature .sample   n_non_na dilution copies_per_swab_med copies_per_swab_mean
   <chr>    <chr>        <int>    <dbl>               <dbl>                <dbl>
 1 C0175A1  06810000…        3        5                   0                    0
 2 C0022A1  06810000…        3        5                   0                    0
 3 C0059E1  06810000…        3        5                   0                    0
 4 UC101    06810000…        3        5                   0                    0
 5 FF00018  06810000…        3        5                   0                    0
 6 FF00051  06810000…        3        5                   0                    0
 7 185329   06810000…        3       20                   0                    0
 8 C0028A1  06810000…        3       20                   0                    0
 9 C0112A1  06810000…        3       20                   0                    0
10 FF00004  06810000…        3       20                   0                    0
# ℹ 310 more rows
# ℹ 19 more variables: copies_per_swab_cv <dbl>, uid <chr>, mg_pid <chr>,
#   mg_visit_code <chr>, qpcr_sample_type <fct>, sample_type <chr>,
#   control_type <chr>, qpcr_sample_id <chr>, vibr_sample_id <chr>,
#   ext_lib_plate_nb <chr>, target <fct>, fluor <chr>, strain_group_qpcr <chr>,
#   taxon_label <chr>, LBP <fct>, strain_id <fct>, strain_origin <fct>,
#   biose_id <chr>, pcr_plate_ids <chr>

Save SummarizedExperiment objects

We first check that the SE_qPCR_agg object is formatted as it should for its integration in the MAE.

# We remove `mg_pid` and `mg_visit_code` from the colData to avoid conflict when merging with the metagenomics data.
colData(SE_qPCR_agg) <- colData(SE_qPCR_agg)[, -which(colnames(colData(SE_qPCR_agg)) %in% c("mg_pid", "mg_visit_code"))]
# We check that the `SE_qPCR_agg` object is formatted as it should
SE_qPCR_agg <- check_se(SE_qPCR_agg)

Save the SE objects to disk

saveRDS(
  SE_qPCR_raw, 
  str_c(
    get_01_output_dir(),  
    "03_se_pcr_raw_", today() |> str_remove_all("-"), ".rds"
    )
  )


saveRDS(
  SE_qPCR_agg, 
  str_c(
    get_01_output_dir(),  
    "03_se_pcr_agg_", today() |> str_remove_all("-"), ".rds"
    )
  )